ABCMdb

ABCG2 p.Cys603Ala

Predicted by SNAP2:	A: D (85%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (91%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Oligomerization of the human ABC transporter ABCG2... Biochemistry. 2005 Aug 16;44(32):10893-904. Bhatia A, Schafer HJ, Hrycyna CA
Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers.
Biochemistry. 2005 Aug 16;44(32):10893-904., 2005-08-16 [PMID:16086592]

Abstract [show]
Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.

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67 ABCG2∆EC-C, which contains all three endogenous extracellular cysteines replaced with alanine (C592A, C603A, C608A), was constructed by site-directed mutagenesis of the parent plasmid pTM1-ABCG2 (R482G). Login to comment

[hide] Identification of intra- and intermolecular disulf... J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17. Henriksen U, Fog JU, Litman T, Gether U
Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2.
J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17., 2005-11-04 [PMID:16107343]

Abstract [show]
ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

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No. Sentence Comment
3 Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Login to comment
4 Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Login to comment
6 In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Login to comment
7 Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/ C608A), the transporter displayed impaired plasma membrane expression and function. Login to comment
104 The resulting constructs (C592A, C603A, and C608A) were stably expressed in HEK293 cells using the bicistronic vector pCIN4 (28), and total cell lysates were analyzed by Western blotting in the presence of increasing concentrations of DTT (Fig. 3). Login to comment
107 Interestingly, the disulfide bridge-linked dimer was completely absent in the C603A mutant (Fig. 3B). Login to comment
113 Subsequent immunocytochemistry analysis of the single mutants was in agreement with the Western blot analysis and supported that the wt and C603A mutant were expressed almost exclusively in the plasma membrane (Fig. 3, B and D). Login to comment
114 In comparison, C592A and C608A exhibited less apparent plasma membrane staining and substantially more intracellular staining than in wt and C603A (Fig. 3, A and C). Login to comment
118 Only in C603A, the resistance FIGURE 3. Login to comment
120 C608A (A), C603A (B),C592A(C),andwt(D)areshown.Immunostainings of the corresponding cells are shown directly below. Login to comment
125 The analysis showed that, although C603A tended to have a higher total expression, there were no significant changes in the expression of C592A and C608A. Login to comment
129 To further explore this hypothesis, we mutated two cysteines at a time, resulting in C592A/C603A, C592A/C608A, and C603A/C608A, each of which contained one remaining extracellular cysteine. Login to comment
133 In contrast, protein expression was very low in the construct containing only Cys-592 (C603A/ C608A) (Figs. 5C and 6C) and slightly lower than wt for C592A/C603A, in which only Cys-608 is present (Figs. 5A and 6C). Login to comment
134 Notably, we also detected dimer formation in C603A/C608A and C592A/C603A. Login to comment
136 A, comparison of cell survival between non-transfected HEK293 cells (E) and HEK293 cells transfected with ABCG2-wt (●), C592A (‚), C603A (ƒ), and C608A (Ⅺ). Login to comment
145 Shown are C592A/C603A (A), wt (B), C603A/C608A (C), and C592A/ C608A (D). Login to comment
152 Staining of the transfected HEK293 cells showed that, although C592A/C608A (Fig. 5D) mostly resembled the membrane-localized expression pattern of wt (Fig. 5B), C592A/C603A displayed largely intracellular staining (Fig. 5A), and C603A/C608A was hardly visible (Fig. 5C). Login to comment
157 For both C603A/C608A and C592A/C603A, we observed a marked decrease in resistance to mitoxantrone and a concomitant decrease in expression (Fig. 6, B and C). Login to comment
160 We also substituted all three extracellular cysteines in ABCG2 simultaneously (C592A/C603A/C608A). Login to comment
162 Specifically, we were almost unable to detect any expressed protein in the immunostainings (Fig. 7B) To further explore the function of the hypothesized disulfide bridges, we performed efflux experiments on the mutants containing either both Cys-592 and Cys-608 (C603A), predicted to form an intramolecular disulfide bridge, or Cys-603 only (C592A/C608A), predicted to form an intermolecular disulfide bridge (Fig. 9). Login to comment
164 C603A displayed efflux similar to wt, whereas efflux in C592A-C608A was slightly decreased (Fig. 9). Login to comment
165 The pattern was, however, different when analyzing another substrate for ABCG2, BODIPY-prazosin; i.e. we observed BODIPY-prazosin efflux similar to the wt in C603A, whereas in C592A-C608A we could not detect any evidence for BODIPY-prazosin efflux (Fig. 9). Login to comment
168 Without prior TCEP reduction, we observed no labeling consistent with no cysteines on the extracellular face of the transporter available for biotinylation (Fig. 10); however, upon TCEP treatment, we found clear biotin labeling of both C603A and C592A/C608A. Login to comment
172 A, comparison of cell survival between empty HEK293 (E) and ABCG2-wt (●), C592A/C603A (‚), C592A/C608A (ƒ), and C603A/C608A (Ⅺ). Login to comment
181 Of all mutants tested, only C603A showed clear staining with anti-ABCG2 (5D3) (Fig. 11); all other mutants showed no staining (Fig. 11). Login to comment
196 The unconstrained plasma membrane targeting of C603A also indicates that we have not disrupted dimerization in the native membrane, because evidence has been obtained in the homologous FIGURE 7. Login to comment
202 The transmitted light image of the C592A/C603A/C608A (3cys) sample is also shown. Login to comment
206 Empty HEK293 (●), ABCG2-wt (Œ), and ABCG2-C592A/C603A/C608A (3cys) (f) are shown. Login to comment
219 In addition, biochemical evidence for the existence of this disulfide bridge was obtained from the biotinylation experiment showing biotinylation in C603A only with prior TCEP reduction (Fig. 10). Login to comment
221 Cys-592 and Cys-608 might nonetheless form intermolecular disulfide bridges when they are "alone" as indicated from the results with the double mutations; i.e. both in C603A/C608A (where Cys-592 is alone) and in C592A/C603A (where Cys-608 is alone), we observed some disulfide bridge-linked dimers despite the fact that Cys603 is mutated. Login to comment
223 Efflux of BODIPY-prazosin and mitoxantrone is not affected by removal of the intermolecular disulfide bridge (C603A), whereas removal of the intramolecular disulfide bridge affects BODIPY-prazosin efflux (C592A/C608A). Login to comment
231 Cells stably expressing wt, C603A, or C592A/C608A ABCG2 were exposed to a cysteine-reactive biotinylation agent after incubation with (ϩ) or without (-) the reducing agent TCEP. Login to comment
241 Mutating all three extracellular cysteines in ABCG2 (C592A/C603A/ C608A) at the same time had detrimental effects on the transporter. Login to comment
243 This supports the conclusion that, although we can disrupt the putative intermolecular symmetrical disulfide bridge involving Cys-603 (C603A mutant) and although we can remove the putative intramolecular disulfide bridge between Cys-592 and Cys-608 (C592A/C608A mutant) without any major impact on expression of the transporter, it is not possible to remove both of them simultaneously. Login to comment

[hide] Role of Cys-603 in dimer/oligomer formation of the... Cancer Sci. 2005 Dec;96(12):866-72. Kage K, Fujita T, Sugimoto Y
Role of Cys-603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2.
Cancer Sci. 2005 Dec;96(12):866-72., [PMID:16367905]

Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) is a half-molecule ATP-binding cassette transporter that we have previously suggested might function as a homodimer, bridged by disulfide bonds. In the present study, we carried out cysteine-scanning mutagenesis, substituting Ser for Cys, and established 12 PA317 transfectants expressing BCRP mutants with possible disruptions to their S-S bonds. Western blot analysis of BCRP from the wild-type transfectants (PA/WT) confirmed that the wild-type protein migrates as a 140-kDa dimer under non-reducing conditions, but as a 70-kDa monomer under reducing conditions. However, under non-reducing conditions the BCRP-C603S mutant migrated both as a 70-kDa monomer and a 140-kDa dimer, whereas all other mutant BCRP migrated only as dimers. PA317 cells transfected with C603S-BCRP (PA/C603S) showed either similar or only marginally lower SN-38 resistance than PA/WT cells, despite the reduced levels of BCRP dimer in these cells. Moreover, the degree of SN-38 resistance in the mutant BCRP transfectants was found to be associated with the monomer expression levels under reducing conditions. Reverse transcription-polymerase chain reaction analysis showed that the BCRP mRNA levels were similar in the transfectants. We subsequently generated six C603X mutants of BCRP (X=D, H, R, Y, A and W) and carried out western blot analysis and drug sensitivity assays. The results were equivalent to those from the PA/C603S cells, with some variations that again corresponded to the monomer levels. Our findings suggest that Cys-603 is an important residue in the covalent bridge between BCRP monomers but that a functioning unit of BCRP may not necessarily require covalent linkages.

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No. Sentence Comment
143 (c) SN-38 sensitivity of parental PA317, PA/WT, PA/C603D, PA/C603Y, PA/ C603S and PA/C603A. Login to comment

[hide] ATP-binding cassette, subfamily G (ABCG family). Pflugers Arch. 2007 Feb;453(5):735-44. Epub 2006 Sep 16. Kusuhara H, Sugiyama Y
ATP-binding cassette, subfamily G (ABCG family).
Pflugers Arch. 2007 Feb;453(5):735-44. Epub 2006 Sep 16., [PMID:16983557]

Abstract [show]
This review summarizes the characteristics of the ATP-binding cassette, subfamily G (ABCG family), which has five members: ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8. The members consist of a single ABC cassette in the amino terminal followed by six putative transmembrane domains, and to become functionally active, they form homo- or obligate heterodimers. Except for ABCG2, the members of the ABCG family play an important role in efflux transport of cholesterol. Mutations causing a loss of function of ABCG5 or ABCG8 are associated with sitosterolemia characterized by accumulation of phyto- and shellfish sterols. Unlike other members, ABCG2 is not involved in cholesterol efflux, but it exhibits broad substrate specificity to xenobiotic compounds. ABCG2 confers cancer cells resistance to anticancer drugs and plays a critical role in the pharmacokinetics of drugs in the clearance organs and tissue barriers. ABCG2 is also associated with a subpopulation phenotype of stem cells. Genetic polymorphisms of ABCG2 have been suggested to account for the interindividual differences in the pharmacokinetics of drugs.

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74 Furthermore, substitution of alanine for cysteine 603 did not affect membrane localization and resistance to mitoxantrone, although it abolished dimerization [24]. Login to comment

[hide] Homology modeling of breast cancer resistance prot... J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15. Hazai E, Bikadi Z
Homology modeling of breast cancer resistance protein (ABCG2).
J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15., [PMID:18249138]

Abstract [show]
BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.

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245 However, in our model, R482 cannot form interaction with rhodamine, but L484 is in interacting distance Table 3 Mutations on BCRP and their effect on its function Mutation Effect/results Reference V12M Did not effect Hemato and MTX transport Tamura et al. (2006) G51C Did not effect Hemato and MTX transport Tamura et al. (2006) K86M Inactivates transporter (dominant negative effect on ATPase activity); alters subcellular distribution Henriksen et al. (2005a) K86M Transporter inactive, but still able to bind ATP Ozvegy et al. (2002) Q126stop Defective porphyrin transport Tamura et al. (2006) Q141K Did not effect Hemato and MTX transport Tamura et al. (2006) T153M Did not effect Hemato and MTX transport Tamura et al. (2006) Q166E Did not effect Hemato and MTX transport Tamura et al. (2006) I206L Did not effect Hemato and MTX transport Tamura et al. (2006) F208S Defective porphyrin transport Tamura et al. (2006) S248P Defective porphyrin transport Tamura et al. (2006) E334stop Defective porphyrin transport Tamura et al. (2006) F431L Effects MTX transport Tamura et al. (2006) S441N Defective porphyrin transport Tamura et al. (2006) E446-mutants No drug resistance Miwa et al. (2003) R482G, R482T Effects MTX transport Tamura et al. (2006) R482T Substrate drug transport and inhibitor efficiency is not mediated by changes in drug-binding Pozza et al. (2006) R482G, R482T Substitution influence the substrate specificity of the transporter Ozvegy et al. (2002) R482G, R482T Altered substrate specificity Honjo et al. (2001) R482G Methotrexate not transported Chen et al. (2003b) Mitomo et al. (2003) R482G Resistance to hydrophilic antifolates in vitro, G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates Shafran et al., (2005) R482G Three distinct drug, binding sites Clark et al. (2006) R482G Altered substrate specificity, granulocyte maturation uneffected Ujhelly et al. (2003) R482 mutants Higher resistance to mitoxantrone and doxorubicin than wt Miwa et al. (2003) R482X Affects substrate transport and ATP hydrolysis but not substrate binding Ejendal et al. (2006) F489L Impaired porphyrin transport Tamura et al. (2006) G553L; G553E Impaired trafficing, expression, and N-linked glycosylation Polgar et al. (2006) L554P Dominant negative effect on drug sensitivity Kage et al. (2002) N557D Resistance to MTX, but decreased transport of SN-38; N557E no change in transport compared to wt Miwa et al. (2003) F571I Did not effect Hemato and MTX transport Tamura et al. (2006) N590Y Did not effect Hemato and MTX transport Tamura et al. (2006) C592A Impaired function and expression Henriksen et al. (2005b) C592A/C608A Restored plasma mb expression; MTX transport normal, BODIPY-prazosin impaired Henriksen et al. (2005b) C603A Disulfide bridge; no functional or membrane targeting change Henriksen et al. (2005b) C608A Impaired function and expression Henriksen et al. (2005b) D620N Did not effect Hemato and MTX transport Tamura et al. (2006) H630X No change in transport Miwa et al. (2003) Cand N-terminal truncated Impaired trafficing Takada et al. (2005) with the ligand. Login to comment

[hide] Effect of cysteine mutagenesis on the function and... J Pharmacol Exp Ther. 2008 Jul;326(1):33-40. Epub 2008 Apr 22. Liu Y, Yang Y, Qi J, Peng H, Zhang JT
Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2.
J Pharmacol Exp Ther. 2008 Jul;326(1):33-40. Epub 2008 Apr 22., [PMID:18430864]

Abstract [show]
ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. Its overexpression causes multidrug resistance in cancer chemotherapy. Based on its apparent half size in sequence when compared with other traditional ABC transporters, ABCG2 has been thought to exist and function as a homodimer linked by intermolecular disulfide bonds. However, recent evidence suggests that ABCG2 may exist as a higher form of oligomers due to noncovalent interactions. In this study, we attempted to create a cysless mutant ABCG2 as a tool for further characterization of this molecule. However, we found that the cysless mutant ABCG2 is well expressed but not functional. Mapping of the cysteine residues showed that three cysteine residues (Cys284, Cys374, and Cys438) are required concurrently for the function of ABCG2 and potentially for intramolecular disulfide bond formation. We also found that the cysteine residues (Cys592, Cys603, and Cys608) in the third extracellular loop are involved in forming intermolecular disulfide bonds and that mutation of these residues does not affect the expression or drug transport activity of human ABCG2. Thus, we conclude that Cys284, Cys374, and Cys438, which may be involved in intramolecular disulfide bond formation, are concurrently required for ABCG2 function, whereas Cys592, Cys603, and Cys608, potentially involved in intermolecular disulfide bond formation, are not required.

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111 Mutation of one or two of these residues (I2-CL, C284A, and C374A) did not signifi- TABLE 1 Primers used for construction of cysless mutants Mutations Primer Sequence RESa C43A TTTCATAACATTGCCTATCGAGTAAAACTGAAG BsrDI C55A GCTTTCTACCTGCACGAAAACCAGTTGAG BsgI C119A GCCAATTTCAAAGCGAATTCAGGTTACGTGG EcoRI C284A GAATCAGCTGGATATCACGCTGAGGCCTATAATAAC EcoRV C374A ACACCACCTCCTTCGCTCATCAACTCAGATG None C438A CTGACGACCAACCAAGCTTTCAGCAGTGTTTC HindIII C491A TATATTTACCGCTATAGTATACTTCATGTTAGG AccI C544A CTTCTCATGACGATCGCTTTTGTGTTTATGATG PvuI C592A GGACAAAACTTCGCCCCGGGACTCAATGCAA SmaI C603A/C608A AGGAAACAATCCTGCTAACTATGCAACAGCTACTGGCGAAGAATATTT -NspI C635A CACGTGGCCTTGGCTGCAATGATTGTTATTTTC BsrDI a Restriction (RES) enzyme digestion sites engineered in the primer for the convenience of detection. Login to comment
112 The primer sequence for the C603A/C608A mutant does not contain the NspI site present in the wild-type sequence. Login to comment
131 To test this hypothesis, we engineered another construct that has all three cysteine residues in the third extracellular loop mutated to alanine (C592A, C603A, and C608A) to determine whether dimers linked by intermolecular disulfide bonds exist with this mutant. Login to comment

[hide] Interaction with the 5D3 monoclonal antibody is re... J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21. Ozvegy-Laczka C, Laczko R, Hegedus C, Litman T, Varady G, Goda K, Hegedus T, Dokholyan NV, Sorrentino BP, Varadi A, Sarkadi B
Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.
J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21., 2008-09-19 [PMID:18644784]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

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7 When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. Login to comment
28 Interestingly, mutation of Cys-603 to Ala, Gly, or Ser does not remarkably influence the expression and functionality of the transporter (9-11). Login to comment
168 Fig. 4A shows that all Cys-to-Ala mutants (except for C603A/ C608A that was expressed in very low amount and exclusively in an underglycosylated form) could be detected by Western blotting, using the ABCG2-specific BXP-21 antibody. Login to comment
169 The mutants C592A and C603A showed expression levels comparable to that of the wild-type ABCG2, whereas the amount of double mutant C592A/C608A or the triple Ala mutant proteins was about 50% of that seen for the wild-type ABCG2. Login to comment
173 Hoechst 33342 and pheophorbide A were transported by all of the mutants (except for C603A/C608A); however, the mitoxantrone transport capacity of the mutants, lacking the intramolecular or both disulfide bonds, was significantly weaker than that of the R482G or C603A variants. Login to comment
174 There was also a difference in rhodamine123 uptake, with the C592A and C592A/ C603A mutants showing practically no transport activity (Fig. 4B). Login to comment
176 When cells expressing the different Cys-to-Ala mutants were labeled with the 5D3 antibody, we found that only the C603A variant had a clearly detectable 5D3 labeling and the C592A/ C608A mutant showed some weak 5D3 binding capacity (Fig. 5A, upper panel). Login to comment
177 Similar to that seen in the case of the wild-type ABCG2, PFA fixation (Fig. 5A, lower panel) or Ko143 treatment (not shown) of the cells expressing the C603A mutant and the double mutant C592A/C608A resulted in an increased 5D3 binding. Login to comment
180 Fig. 5B shows that all mutants could be detected by the BXP-21 antibody, recognizing an intracellular epitope of ABCG2, and all of them were present in the plasma membrane (except for the hardly expressed C603A/C608A double mutant, which was found, for the most part, intracellularly). Login to comment
181 However, 5D3 labeling analyzed by confocal microscopy gave the same result as the flow cytometry measurements, that is, only the cells expressing the wild-type ABCG2, C603A, and the C592A/ C608A variants (the latter one seen only at increased detector voltage) could bind the 5D3 antibody. Login to comment
184 We found that, in contrast to a 30-40% inhibition found in the case of the wild-type ABCG2, 5D3 did not influence the Hoechst 33342 transport activity of the C592A/C608A and C592A/C603A/C608A mutants (data not shown). Login to comment
186 Effect of DTT on 5D3 Labeling of the Cys-to-Ala Mutants-To test whether decreased 5D3 binding in ABCG2-expressing cells treated with DTT was due to the reduction of the extracellular cysteines, we also examined the effect of DTT on 5D3 labeling of the mutants C603A and C592A/C608A in native or PFA-fixed cells. Login to comment
187 Fig. 6, A and B, shows that DTT is still effective in the reduction of 5D3 binding in the case of the C603A mutant but has practically no effect on 5D3 labeling of the C592A/C608A mutant. Login to comment
199 Interaction of ABCG2 with the 5D3 Monoclonal Antibody SEPTEMBER 19, 200•VOLUME 283•NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 26065 in 5D3 binding that reached its minimum (almost the fluorescence of the background) at 10-50 mM DTT in ABCG2 and C603A-expressing cells, whereas DTT had no effect on labeling of the C592A/C608A double mutant (Fig. 6C). Login to comment
234 To find out which disulfide bridge is important for epitope formation, we analyzed different Cys-to-Ala mutants lacking the intermolecular (C603A), the intramolecular (C592A, C608A, C592A/C608A), or both kinds of (C592A/C603A, C603A/C608A, or C592A/ C603A/C608A) S-S bonds. Login to comment
235 We found that the C603A mutant behaves similarly to ABCG2 having intact S-S bridges. Login to comment
236 The C603A mutant, which can be expressed in an amount comparable to ABCG2, is found in the plasma membrane (Figs. Login to comment
249 The single mutants, lacking the intramolecular S-S bond, i.e. C592A, C608A, as well as the C592A/C603A/C608A variant, had clearly detectable expression levels, were present in the plasma membrane, and were functional for active transport with somewhat altered substrate specificities (Figs. 4 and 5). Login to comment

[hide] Fluorescence resonance energy transfer (FRET) anal... Int J Biochem Mol Biol. 2010;1(1):1-11. Ni Z, Mark ME, Cai X, Mao Q
Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells.
Int J Biochem Mol Biol. 2010;1(1):1-11., [PMID:20622991]

Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein and expressed in HEK293 cells by transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by immunoblotting, 5D3 antibody binding, and flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form a dimer/oligomer in vivo in intact cells using the FRET technique. We have also shown that Cys(603) alone does not seem to be essential for dimer/oligomer formation of BCRP.

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4 Wild-type BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein and expressed in HEK293 cells by transient transfection. Login to comment
7 Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. Login to comment
8 C603A was predominantly expressed in the plasma membrane as was wild-type protein. Login to comment
9 Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Login to comment
38 In the present study, we used FRET microscopy to elucidate dimer/oligomer formation of BCRP in HEK293 cells, a method that avoids artificial alterations (e.g., formation or breakdown of disulfide bonds) during biochemical sample preparation. To further understand whether Cys603 plays an important role in dimer/oligomer formation of BCRP, we generated the mutant C603A and investigated the effect of the mutation of Cys603 on dimer/oligomer formation and activity of BCRP. Login to comment
54 Construction of CFP- or YFP-tagged wild-type BCRP or C603A The full length BCRP cDNA fragment was amplified using the pcDNA3.1 plasmid containing full length wild-type BCRP cDNA as a template with a forward primer containing a XhoI site (5`-CTTCGGCTCGAGCTATGTCTTCCAGTAATGTCGAA G-3`) and a reverse primer (5' GTCAGAATCTAGACAAGCTTGGTACCGAGCTCGGATCC-3'). Login to comment
56 The resultant YFP-BCRP plasmid was used as a template for PCR mutagenesis to generate the C603A mutant in which Cys603 was replaced with Ala. Login to comment
57 The primers used to generate C603A were 5`- GCAACAGGAAACAATCCTGCTAACTATGCAACATGTAC-3` and 5`- GTACATGTTGCATAGTTAGCAGGATTGTTTCCTGTTGC-3`. Login to comment
58 To generate CFP-tagged wild-type BCRP or C603A, the YFP-BCRP plasmids were digested with XhoI and BamHI, and the DNA fragments containing wild-type and mutant BCRP cDNAs were subcloned into the pECFP-C1 plasmid. Login to comment
60 In these constructs, CFP or YFP was attached to the N-terminus of wild-type BCRP or C603A. Login to comment
65 In this 4 µg of cDNA, an equal amount of CFP- and YFP-tagged wild-type BCRP or C603A cDNAs were used at a 1:1 ratio. Login to comment
73 Detection of cell surface expression of wild-type BCRP or C603A The 5D3 antibody recognizes a conformation-sensitive extracellular epitope in BCRP [32]. Login to comment
74 Binding of the 5D3 antibody to the surface of cells expressing wild-type BCRP or C603A was examined using flow cytometry as previously described [33]. Login to comment
79 Flow cytometric efflux assay MX efflux activity of wild-type BCRP or C603A was determined using flow cytometry as previously described [22]. Login to comment
82 Then, MX efflux activity of cells expressing wild-type BCRP or C603A was determined as previously described [22]. Login to comment
85 FRET microscopy and data analysis HEK293 cells were grown in polystyrene vessels and transiently co-transfected with a total of 1 µg of CFP- and YFP-tagged wild-type BCRP or C603A cDNAs or the CFP/YFP control vectors at a 1:1 ratio as described above. Login to comment
107 Results Expression of wild-type BCRP and C603A in HEK293 cells An equal amount of CFP- and YFP-tagged wild-type BCRP or C603A cDNAs or the CFP/ YFP control vectors were co-transfected into HEK293 cells, and the expression levels of wild-type and mutant BCRP were examined by immunobloting of whole cell lysates. Login to comment
108 The expression levels of wild-type BCRP and C603A were comparable (Figure 1A), indicating that the expression and/or stability of BCRP was not affected by the mutation of Cys603. Login to comment
110 Cell surface expression of wild-type BCRP and C603A We next determined cell surface expression of wild-type BCRP and C603A by measuring binding of the phycoerythrin-conjugated 5D3 to the surface of cells expressing these proteins. Login to comment
112 However, cells expressing wild-type BCRP or C603A demonstrated a significant shift in phycoerythrin fluorescence between the 5D3 and IgG2b treatments (Figure 1B), supporting cell surface expression of wild-type BCRP or C603A, respectively. Login to comment
113 Cellular localization of wild-type BCRP or C603A To determine whether the mutation of Cys603 could affect the plasma membrane localization of BCRP, transfected cells were analyzed using confocal fluorescence microscopy. Login to comment
116 Thus, in the case of wild-type BCRP or C603A, a strong CFP or YFP fluorescence signal or the signal in the merged image was observed primarily on the plasma membrane (Figure 2), suggesting that wild-type BCRP or C603A was predominantly targeted to the plasma membrane of HEK293 cells. Login to comment
117 MX efflux activity of wild-type BCRP or C603A We next determined if the mutation of Cys603 affects BCRP efflux activity using a model fluorescent substrate MX. Login to comment
118 Incubation of cells expressing wild-type BCRP or C603A with the BCRP-specific inhibitor FTC resulted in a similarly significant increase in the intracellular MX accumulation compared to that without FTC treatment (data not shown), suggesting that MX is actively effluxed by wild-type BCRP or C603A. Login to comment
119 Thus, the FTC-inhibitable MX efflux activities of wild-type BCRP and C603A were comparable (Figure 3). Login to comment
120 The FTC-inhibitable MX efflux activities of cells expressing only the CFP/YFP pair control were significantly lower than those of the cells expressing wild-type BCRP or C603A (Figure 3). Login to comment
121 These results suggest that C603A fully retains MX efflux activity comparable to wild-type protein. Login to comment
122 Detection of dimer/oligomer formation of wild-type BCRP or C603A by FRET analysis We then determined the effect of the mutation of Cys603 on dimer/oligmer formation of BCRP in intact cells using FRET microscopy. Login to comment
123 FRET analysis was performed for cells expressing the CFP/YFP pair control, the CFP/YFP-tagged wild-type BCRP or C603A, or CFP alone. Login to comment
130 The FRET efficiencies of cells expressing CFP/YFP-tagged C603A were comparable to those of cells expressing CFP/YFP-tagged wild-type BCRP, suggesting that C603A retained the same ability to form a dimer/ oligomer as wild-type protein. Login to comment
131 Chemical cross-linking To further confirm if the monomers of wild-type BCRP or C603A exist in close proximity in intact cells, we performed chemical cross-linking experiments using the homobifunctional amine-reactive cross-linker DSS (11.4 Å arm length) on intact cells expressing these proteins. Login to comment
132 Either wild-type BCRP or C603A could be cross-linked as indicated by the appearance of higher molecule mass bands corresponding to dimers and oligomers of the 95 kDa CFP/YFP-tagged BCRP or C603A (Figure 5). Login to comment
135 This approach allows us to elucidate dimer/ oligomer formation of BCRP in a native cellular membrane environment without the need of biochemical sample preparation. To perform the FRET assay, the fluorescent protein CFP or YFP was attached to the N-terminal end of wild-type BCRP or the mutant C603A. Login to comment
142 With this method, we estimated the FRET efficiencies of intact cells expressing CFP/YFP-tagged wild-type BCRP or C603A, the CFP/YFP pair control or CFP alone. Login to comment
148 Ala substitution of Cys603 did not deteriorate expression, plasma membrane localization, and activity of BCRP in HEK293 cells (Figs. 1 - 3), which is in good agreement with previous studies [18,24,25]. Login to comment
150 This conclusion was obtained by the demonstration that wild-type BCRP migrated as a band of approximately double the size of a BCRP monomer under non-reducing conditions; however, after Cys603 was mutated to Ala, the C603A mutant migrated as a single monomeric band [18,23,24]. Login to comment
151 Nevertheless, at least one study [23] showed that a substantial amount of C603A and other mutants at position Cys603 remained as dimers under non-reducing conditions. It is worth noting that the observation of dimer/oligomer formation of BCRP via intermolecular disulfide bonds has so far been reported all using in vitro biochemical methods such as immunoblotting under nonreducing conditions. It is possible that oxidation during biochemical sample preparation may cause formation of intermolecular disulfide bridges or disulfide bonds may be disrupted in the process of cell lysis or sample preparation involving solubilization of membrane proteins with detergents. Login to comment
153 We found that the FRET efficiency of CFP/YFP-tagged C603A was the same as that of CFP/YFP-tagged wild-type BCRP (Figure 4), suggesting that Ala substitution of Cys603 does not affect the ability of BCRP to form a dimer/oligomer. Login to comment
154 Chemical cross-linking experiments also indicate that CFP/YFP-tagged C603A molecules may exist in cells in close proximity (Figure 5). Login to comment
155 Given the fact that C603A retained full expression, plasma membrane targeting, and MX efflux activity comparable to wild-type protein (Figure 1-3), we would argue that the intermolecular disulfide bond formed by Cys603 alone may not be essential for dimerization or oligomerization of BCRP in vivo. Login to comment
12 Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. Login to comment
13 C603A was predominantly expressed in the plasma membrane as was wild-type protein. Login to comment
14 Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/ oligmer formation as wild-type BCRP. Login to comment
33 In the present study, we used FRET microscopy to elucidate dimer/oligomer formation of BCRP in HEK293 cells, a method that avoids artificial alterations (e.g., formation or breakdown of disulfide bonds) during biochemical sample preparation. To further understand whether Cys603 plays an important role in dimer/oligomer formation of BCRP, we generated the mutant C603A and investigated the effect of the mutation of Cys603 on dimer/oligomer formation and activity of BCRP. Login to comment
49 Construction of CFP- or YFP-tagged wild-type BCRP or C603A The full length BCRP cDNA fragment was amplified using the pcDNA3.1 plasmid containing full length wild-type BCRP cDNA as a template with a forward primer containing a XhoI site (5`- CTTCGGCTCGAGCTATGTCTTCCAGTAATGTCGAA G-3`) and a reverse primer (5' GTCAGAATCTA- GACAAGCTTGGTACCGAGCTCGGATCC-3'). Login to comment
51 The resultant YFP-BCRP plasmid was used as a template for PCR mutagenesis to generate the C603A mutant in which Cys603 was replaced with Ala. Login to comment
52 The primers used to generate C603A were 5`-GCAACAGGAAACAATCCTGCTAACTATGCA ACATGTAC-3` and 5`-GTACATGTTGCATAGTTAGCA GGATTGTTTCCTGTTGC-3`. Login to comment
53 To generate CFP-tagged wild-type BCRP or C603A, the YFP-BCRP plasmids were digested with XhoI and BamHI, and the DNA fragments containing wild-type and mutant BCRP cDNAs were subcloned into the pECFP-C1 plasmid. Login to comment
55 In these constructs, CFP or YFP was attached to the N-terminus of wild-type BCRP or C603A. Login to comment
68 Detection of cell surface expression of wild-type BCRP or C603A The 5D3 antibody recognizes a conformation-sensitive extracellular epitope in BCRP [32]. Login to comment
69 Binding of the 5D3 antibody to the surface of cells expressing wild-type BCRP or C603A was examined using flow cytometry as previously described [33]. Login to comment
77 Then, MX efflux activity of cells expressing wild-type BCRP or C603A was determined as previously described [22]. Login to comment
80 FRET microscopy and data analysis HEK293 cells were grown in polystyrene vessels and transiently co-transfected with a total of 1 µg of CFP- and YFP-tagged wild-type BCRP or C603A cDNAs or the CFP/YFP control vectors at a 1:1 ratio as described above. Login to comment
102 Results Expression of wild-type BCRP and C603A in HEK293 cells An equal amount of CFP- and YFP-tagged wild-type BCRP or C603A cDNAs or the CFP/YFP control vectors were co-transfected into HEK293 cells, and the expression levels of wild-type and mutant BCRP were examined by immunobloting of whole cell lysates. Login to comment
103 The expression levels of wild-type BCRP and C603A were comparable (Figure 1A), indicating that the expression and/ or stability of BCRP was not affected by the mutation of Cys603. Login to comment
105 Cell surface expression of wild-type BCRP and C603A We next determined cell surface expression of wild-type BCRP and C603A by measuring binding of the phycoerythrin-conjugated 5D3 to the surface of cells expressing these proteins. Login to comment
109 A, whole cell lysates (20 μg of protein each lane) prepared from HEK293 cells transiently transfected with CFP/YFP-tagged wild-type BCRP or C603A cDNA constructs or the CFP/YFP control vectors were immunoblotted as described. Login to comment
115 The histograms for CFP/YFP, wild-type BCRP, and C603A are indicated above the graphs. Login to comment
125 Confocal fluorescence images of HEK293 cells co-transfected with CFP/YFP-tagged wild-type BCRP or C603A cDNAs. HEK293 cells were co-transfected with CFP/YFP-tagged wild-type BCRP or C603A cDNA constructs or the CFP/YFP control vectors and processed for confocal fluorescence microscope analysis as described. Login to comment
127 Images for CFP/YFP, wild-type BCRP, and C603A are indicated on the left. Login to comment
128 the cells expressing wild-type BCRP or C603A (Figure 3). Login to comment
129 These results suggest that C603A fully retains MX efflux activity comparable to wild-type protein. Login to comment
138 The FRET efficiencies of cells expressing CFP/ YFP-tagged C603A were comparable to those of cells expressing CFP/YFP-tagged wild-type BCRP, suggesting that C603A retained the same ability to form a dimer/oligomer as wild-type protein. Login to comment
139 Chemical cross-linking To further confirm if the monomers of wild-type BCRP or C603A exist in close proximity in intact cells, we performed chemical cross-linking experiments using the homobifunctional amine-reactive cross-linker DSS (11.4 Å arm length) on intact cells expressing these proteins. Login to comment
140 Either wild-type BCRP or C603A could be cross-linked as indicated by the appearance of higher mole- Figure 3. Login to comment
141 FTC-inhibitable MX efflux activities of wild-type BCRP and C603A. Login to comment
147 FRET efficiencies of cells co-transfected with CFP/YFP-tagged wild-type BCRP or C603A cDNAs. HEK293 cells were co-transfected with CFP/ YFP-tagged wild-type BCRP or C603A cDNA constructs or the CFP/YFP control vectors or the CFP vector alone and processed for determining FRET efficiencies as described. Login to comment
160 With this method, we estimated the FRET efficiencies of intact cells expressing CFP/YFP-tagged wild-type BCRP or C603A, the CFP/YFP pair control or CFP alone. Login to comment
166 Ala substitution of Cys603 did not deteriorate expression, plasma membrane localization, and activity of BCRP in HEK293 cells (Figs. 1 - 3), which is in good agreement with previous studies [18, 24, 25]. Login to comment
168 This conclusion was obtained by the demonstration that wild-type BCRP migrated as a band of approximately double the size of a BCRP monomer under non-reducing conditions; however, after Cys603 was mutated to Ala, the C603A mutant migrated as a single monomeric band [18, 23, 24]. Login to comment
171 Chemical cross-linking of wild-type BCRP or C603A on intact cells using DSS was described in the Materials and Methods section. Login to comment
172 Whole cell lysates prepared from HEK293 cells transiently transfected with CFP/YFP-tagged wild-type BCRP or C603A cDNA constructs or the CFP/YFP control vectors were immunoblotted using the mAb BXP-21 as described. Login to comment
173 The constructs (CFP/YFP, wild-type BCRP, and C603A) are indicated below the blot. Login to comment
174 As indicated with arrows, the molecular weight of CFP/YFP-tagged wild-type BCRP or C603A monomer is approximately 95 kDa. Login to comment
175 substantial amount of C603A and other mutants at position Cys603 remained as dimers under non-reducing conditions. It is worth noting that the observation of dimer/oligomer formation of BCRP via intermolecular disulfide bonds has so far been reported all using in vitro biochemical methods such as immunoblotting under non-reducing conditions. It is possible that oxidation during biochemical sample preparation may cause formation of intermolecular disulfide bridges or disulfide bonds may be disrupted in the process of cell lysis or sample preparation involving solubilization of membrane proteins with detergents. Login to comment
177 We found that the FRET efficiency of CFP/YFP-tagged C603A was the same as that of CFP/YFP -tagged wild-type BCRP (Figure 4), suggesting that Ala substitution of Cys603 does not affect the ability of BCRP to form a dimer/oligomer. Login to comment
178 Chemical cross-linking experiments also indicate that CFP/YFP-tagged C603A molecules may exist in cells in close proximity (Figure 5). Login to comment
179 Given the fact that C603A retained full expression, plasma membrane targeting, and MX efflux activity comparable to wild-type protein (Figure 1-3), we would argue that the intermolecular disulfide bond formed by Cys603 alone may not be essential for dimerization or oligomerization of BCRP in vivo. Login to comment

[hide] ABCG2 transports and transfers heme to albumin thr... J Biol Chem. 2010 Oct 22;285(43):33123-33. Epub 2010 Aug 12. Desuzinges-Mandon E, Arnaud O, Martinez L, Huche F, Di Pietro A, Falson P
ABCG2 transports and transfers heme to albumin through its large extracellular loop.
J Biol Chem. 2010 Oct 22;285(43):33123-33. Epub 2010 Aug 12., 2010-10-22 [PMID:20705604]

Abstract [show]
ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 mum, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 mum.

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6 Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. Login to comment
70 The single-point mutations H598A, C603A, and Y608A were introduced by site-directed mutagenesis using the corresponding primers displayed in Table 1. Login to comment
220 Single-point Mutations H583A and C603A of ECL3 Alter Hemin Binding-We noticed that ECL3 displays a significant sequence similarity with the heme-binding domain of cytochrome b5, with 14.3% identity and 15.6% similarity, as shown in Fig. 6A. Login to comment
224 As shown, the most striking differences were observed for hemin binding because the H583A and C603A mutations lowered the binding affinity 5-6-fold. Login to comment
245 B, dissociation constants for hemin and PPIX binding to H583A, C603A, and Y605A mutant ECL3. Login to comment
278 Heme and hemin appear to be the preferred ligands, because (i) they display the highest binding affinities for both the isolated ECL3 domain (0.5-1 ␮M) and the full-length ABCG2 transporter (0.2 ␮M); (ii) the H583A and C603A single-point mutations alter more markedly the binding of hemin than that of metal-free corresponding PPIX; and (iii) pheophorbide a, which is transported by ABCG2 (26), displays a low binding affinity for ECL3 (3 ␮M), which is even lower for the full protein (Ͼ8 ␮M) when saturated with MTX or doxorubicin, suggesting that pheophorbide a will not bind to ECL3 under physiological conditions. Login to comment

[hide] Structure and function of the human breast cancer ... Curr Drug Metab. 2010 Sep;11(7):603-17. Ni Z, Bikadi Z, Rosenberg MF, Mao Q
Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).
Curr Drug Metab. 2010 Sep;11(7):603-17., [PMID:20812902]

Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.

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138 The recent study using FRET and chemical cross-linking also revealed that Ala substitution of Cys603 had no any effect on dimer/oligomer formation of BCRP in vivo in intact cells [80]. Login to comment
310 Ala substitution of Cys603 led to migration of BCRP bands to a size corresponding to BCRP monomers, but had no effect on plasma membrane expression and function, suggesting that Cys603 participates in intermolecular disulfide bond formation. Login to comment

[hide] Dimerization of ABCG2 analysed by bimolecular fluo... PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3. Haider AJ, Briggs D, Self TJ, Chilvers HL, Holliday ND, Kerr ID
Dimerization of ABCG2 analysed by bimolecular fluorescence complementation.
PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3., [PMID:21991363]

Abstract [show]
ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.

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86 Primer Sequence 59-39 Restriction site Purpose EYFPG2F CCTGTATTTTCAGGAATTCTATGTCTTCCAG EcoRI Generation of ABCG2 tagged N-terminally with complete eYFP YFPG2R GCTTGGTACCGATCTAGAATCCAATTTAAGAATA XbaI Common reverse primer for tagging ABCG2 N-terminally with fragments of vYFP or with complete eYFP VYFPG2F CCTGTATTTTCAGGAATTCATGTCTTCCAG EcoRI Generation of ABCG2 tagged N-terminally with vYFP fragments G2YFPF CCTGTATTTTCAGGGATCCATGTCTTCCAG BamHI Generation of ABCG2 with C-terminal vYFP fragments G2YFPR GAGCTCGGATCCCTCGAGAGAATATTTTTTAAG XhoI Generation of ABCG2 with C-terminal vYFP fragments E211QF ATCTTGTTCTTGGATCAACCTACAACAGGCTTAGACTCAAG n/a Mutating E211Q C603AF ACAGGAAACAATCCGGCCAACTATGCAACATGTACT n/a Mutating C603A doi:10.1371/journal.pone.0025818.t001 USA, [23]). Login to comment
151 A second mutation, C603A, was introduced to prevent the inter-dimer disulphide bond that has been shown to be necessary for homodimer formation on non-denaturing SDS-PAGE, but which is not required for function [13,15]. Login to comment
153 All three constructs (WT, E211Q and C603A) showed BiFC (Figure 6B-D respectively) producing a fluorescence signal far in excess of the background observed with a single transfection (Figure 6A). Login to comment

[hide] Determinants of the activity and substrate recogni... Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18. Szafraniec MJ, Szczygiel M, Urbanska K, Fiedor L
Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).
Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18., [PMID:25036722]

Abstract [show]
The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.

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226 The substitution of Cys603 by Ala caused the transporter to migrate electrophoretically as a monomer, which is not the case when substituting Cys592 or Cys608 by Ala. Login to comment